Capillary electrophoresis (CF) has become a technique of great interest for the separation and analysis of many kinds of ionic species, including inorganic ions, biological mixtures such as proteins, peptides and nucleic acids, detergents, organic acids and many compounds of pharmaceutical nature. Capillary electrophoresis offers the benefits of high resolving power, rapid separations, ability to analyze very small volumes of sample and a desirable simplicity from the point of view of the apparatus required when compared to competing analytical techniques such as liquid chromatography.
The benefits of capillary electrophoresis mentioned above derive to a large extent from the use of narrow diameter capillary tubes in the practice of capillary electrophoresis. The narrow diameter tubes permit efficient removal of the heat generated in the separation process and prevent convective mixing which would degrade the separating power. The narrow diameter tubes also allow high voltages to be used to generate the electric field in the capillary while limiting current flow and hence heat generation. The electric field in the capillary tube which provides the driving force for the separation is produced by the application of high voltage power to the ends of the capillary. The high voltage power is generally applied from a high voltage power supply operating at an operator selected constant voltage during the separation.
Variations on the application of constant voltage operation have been proposed in the past. For example, in order to overcome drift in certain detectors used in CE, Morris et al applied an AC voltage superimposed on a constant DC voltage to drive a CE separation, as described in U.S. Pat. No. 4,909,919. The AC voltage modulates the velocity of the migrating sample components and the detector signal is synchronously demodulated to cancel out drift due for example to temperature fluctuations in the capillary. Likewise, in applications involving the separation of DNA fragments in gel containing capillaries, the high voltage can be applied in a pulsed manner as described in U.S. Pat. No. 5,122,248. The pulsing of the applied voltage results in a greater degree of resolution of the closely related fragments, due to reorientation of analyte molecules in the separating medium during different phases of each pulse. However, in each of these illustrated examples, the average DC level of the output voltage from the power supply remains constant throughout the analysis.
In other cases, it may be preferable to operate the high voltage power supply at a selected level of current through the capillary tube during the entire separation. This is referred to as constant current operation. Takao Tsuda describes certain advantages of operating at a constant current in an article in the Journal of Liquid Chromatography, volume 12 (1989) page 2501, primarily related to the fact that the separation is less dependent on the temperature of the capillary in constant current operation. Similarly, constant power operation is also possible where the level of power dissipated in the capillary is held constant at a selected level.
The separation that occurs in capillary electrophoresis is generally monitored somewhere along the length of the capillary tube by a detector that responds with a signal that is proportional to the concentration at the monitoring point of the analyte(s) within the sample to be measured. Absorbance detectors are commonly used but other kinds of detectors are also possible, such as fluorescence, conductivity, electrochemical and the like. In some cases, the separating medium is modified prior to detection by the addition of reagents to label the analyte or by modifying the conductivity of the medium through ion exchange.
The detectors employed are to a greater or lesser extent non-specific. That is, they respond with an indication of the concentration of many different analytes thus not directly determining the identity of the analyte. In some cases, additional information is available to aid in identifying the sample components, such as when the detector is capable of measuring absorbance at multiple wavelengths so as to obtain an absorbance spectrum or ratios of absorbances at several wavelengths. Alternatively, the separated analytes can be collected as they emerge from the capillary and subjected to further analysis by other techniques. However, multiwavelength detection adds cost and complexity and spectral information may not be available if the analyte is transparent over the spectral range being measured and is being determined instead by an indirect absorbance measurement where the response is due to the displacement of an absorbing species in the separating medium. Post separation analysis by other techniques requires additional time and effort and is not always possible due to the small amount of sample analyzed by capillary electrophoresis.
The detector also permits the measurement of the migration time for each analyte and the migration time can be used to characterize each component of the sample mixture. For purposes herein, the migration time for each analyte is defined as the time period from the start of the analysis to the appearance at the detector of the concentration peak of that analyte. The migration time of each sample component can be compared to the migration times of known standards, and when combined with other knowledge about the origin and character of the sample, allow the identity of the sample components to be inferred. In order for the migration time to be useful, each analyte must appear in the separation at its characteristic migration time over the range of experimental conditions that the analysis is to be used for. In particular, the migration time of each analyte must remain essentially constant over the range of sample compositions that are likely to be encountered.
It is known that the migration time of each analyte tends to fluctuate depending on the composition of the sample. Petr Gebauer, Wolfgang Thormann and Petr Bocek provide a theoretical explanation of how such fluctuations can occur in an article in the Journal of Chromatography, volume 608 (1992) pp. 47-57. This article describes how the concentration of a major component in the sample can affect the migration time of minor sample components by amounts in the tens of percent and cautions that the record of each analysis must be evaluated with great care to avoid making a false determination of the identity of the analytes in the sample. However, the authors do not propose solutions to prevent migration times from varying. Also, neither the patent cited above concerning the application of pulsed voltage nor that describing the imposition of an AC voltage on the DC separation voltage addressess the problem of migration time variation caused by changing sample composition.
In certain applications, the effect of sample composition changes can be magnified, as when it is desired to maximize the sensitivity of a capillary electrophoresis analysis. For example, it may be advantageous for the sample mixture to be analyzed to be dissolved in pure water or other very low conductivity medium rather than, for example, in the separating medium. This causes a phenomenon known as stacking to occur and permits a larger volume of sample mixture to be introduced into the capillary without undesirable broadening of the analyte concentration peaks. However, when the sample is dissolved in pure water to achieve this desirable enhancement in sensitivity, the migration time of the analytes becomes even more dependent on the sample composition.
It would thus be desirable to be able to use migration time as an identifying characteristic for sample components in CE and it would also be desirable to do this while also using the sensitivity enhancement that results from having the sample mixture dissolved in pure water or other low conductivity medium.